INTEGR. COMP. BIOL., 45:61–71 (2005)
Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and
Heavy Metal in Caenorhabditis elegans1
APOLONIA NOVILLO, SEUNG-JAE WON, CHRISTINE LI,
AND IAN
P. CALLARD2
Department of Biology, Boston University, Boston, Massachusetts 02215
SYNOPSIS.
To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C.
elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously
showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene
expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg),
cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were
interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins
and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of
gene expression after progesterone (1025, 1027 M), estradiol (1025 M), cholesterol (1029 M) and cadmium
(0.1, 1 and 10 mM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in
C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after
exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only
estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety
of exogenous steroids, which regulate important metabolic and developmental pathways. The response of
the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable
precursor to signaling molecules that may interact with NRs and we focused on expression of genes related
to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond
principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of
these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.
INTRODUCTION
Nuclear receptors (NRs) are a large superfamily of
transcriptional regulators exclusively found in metazoans (The Arabidopsis Genome Initiative, 2000). Nuclear receptors can be identified on the basis of their
well-conserved DNA binding domain (DBD), which
comprises two Cys2-Cys2 zinc-coordinating modules,
and on the basis of a less conserved domain, called
the ligand binding-domain (LBD), located at the Cterminal. This domain participates in ligand binding,
homo- and heterodimerization, and transcriptional regulation (see review; Freedman, 1997). These proteins
are involved in embryonic pattern formation, development and differentiation of multiple tissue types, sex
determination, metamorphosis, fertility, regulation of
cellular metabolism and homeostasis (Giguere, 1999;
Miyabayashi et al., 1999; Owen and Zelent, 2000; Sluder and Maina, 2001; Enmark and Gustafsson, 2001;
Maglich et al., 2001; Sullivan and Thummel, 2003;
Francis et al., 2003). In addition, NRs are implicated
in diseases such as cancer, diabetes or hormone resistance syndromes, and are extensively investigated for
drug development. More recently, the identification in
the environment of many small molecules (xenobiot-
ics) that bind these NRs has led to the recognition of
the phenomenon of endocrine disruption (ED), and it
is important to understand the effects of xenobiotic
exposure on receptors and potential in vivo signal
transduction pathways. Of particular interest are xenoestrogens because of the broad (pleiotropic) effects
of estrogens and their analogues in all vertebrates.
In the last ten years, complete genome sequences
have become available for C. elegans, C. briggsae
(nematodes), Drosophila melanogaster (insect), Saccharomyces cerevisiae (yeast), Fugu rubripes (teleost
fish), human and mouse. Several authors have compared these genomes, focusing on the NR family (Enmark and Gustafsson, 2001; Sluder and Maina, 2001,
Maglich et al., 2001, 2003) with surprising results.
Thus, dramatically different numbers of NR genes
have been described: 284 in C. elegans, versus only
21 in Drosophila, 68 in fugu and approximately 50 in
humans (Sullivan and Thummel, 2003; Maglich et al.,
2003; Gissendanner et al., 2004). Phylogenetic analysis of the vertebrate NRs, based on the human genome, showed seven groups (0 to VI) of nuclear receptors, with several subgroups in each group (Laudet,
1997; NucleaRDB: http://www.receptors.org/NR/,
Horn et al., 2001). In C. elegans, fifteen of the NR
genes have apparent homologs in insects and vertebrates, and can be assigned to 5 of the large groups of
NR. However, the other NRs in C. elegans cannot be
placed into any of the seven major NR subfamilies and
are labeled ‘‘divergent nematode NRs’’ (Sluder and
1 From the Symposium EcoPhysiology and Conservation: The
Contribution of Endocrinology and Immunology presented at the
Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: [email protected]
61
62
A. NOVILLO
Maina, 2001). Despite the large number of NRs in the
C. elegans genome, the steroid hormone receptor
(NR3) group is not represented in this species. In contrast, in Drosophila at least one member (dERR) of
the NR3 subfamily has been described, confirming the
ancient metazoan origin of this family. Recently, and
consistent with this, the isolation of an estrogen receptor ortholog from a representative group of Protostomes, the mollusk Aplysia californica has been described (Thornton et al., 2003). After reconstruction,
synthesis, and experimental characterization of the
functional domains of this ancestral ER ortholog, the
authors suggest that this gene was lost in the Ecdysozoan (Nematode and Arthropod) lineage. The finding of the ‘ER’ ortholog in a protostome (mollusk)
suggests that several classes of invertebrates may be
subject to endocrine disruption through xenoestrogen
activated gene pathways and networks as in vertebrates, and will explain the observed effects after estradiol exposure in some invertebrate species (see Di
Cosmo et al., 2002; Osada et al., 2003). However, other signal transduction pathways, possibly involving invertebrate receptors not immediately identified as homologs of vertebrate nuclear receptors for estrogens
and xenoestrogens, remain to be identified.
In our prior studies (Custodia et al., 2001) of C.
elegans responses to vertebrate steroids (estradiol, progesterone and testosterone), using vitellogenin as biomarker, we examined the possibility that this end
point, the prototypical estrogen response for all vertebrates except mammals, might be responsive to estradiol in C. elegans. These studies showed a clear
dose response relationship between vitellogenin synthesis and estradiol exposure in culture. Further, using
DNA microarrays, we showed that at least two C. elegans vitellogenin genes (vit-2 and vit-6) were significantly up regulated by estradiol, in a manner reminiscent of the effect of the hormone in vertebrates. Thus,
the possibility exists that estrogens, xenoestrogens and
other related steroids/sterols may be involved in the
regulation of this important metabolic pathway in both
invertebrates and vertebrates. Furthermore, heavy metals, such as cadmium, are known to interact with steroid transcription factors (such as GR and ER) and
have been shown to block/antagonize estrogen action
(Simons et al., 1990; Stoica et al., 2000). It is possible
that responses of C. elegans to cadmium may provide
more information about genomic effects of this metal.
In this paper, we further examine C. elegans NR
genes that may be associated with the up-regulation of
vtg genes. It is possible (likely?) that an incipient network of genes that can be activated by internal and
external environmental signaling via (xeno)estrogens
exists and is associated with this important metabolic
pathway and others at this level of animal organization. Preliminary characterization of the NRs family
proteins in C. elegans and related species (C. briggsae,
B. malayi) suggests that high numbers of NR genes
are expressed (Sluder et al., 1999), and are functional
(Miyabayashi et al., 1999; Kim, 2001). However, all
ET AL.
nematode nuclear receptors belong to the orphan class
of NRs, since ligands have not yet been identified.
DNA sequence analysis suggests that NRs have the
structural potential for ligand binding. In order to explain the unprecedented abundance and diversity of C.
elegans divergent NR genes, confirmed in other nematode genomes (C. briggsae, B. malayi) (Sluder and
Maina, 2001), several related hypotheses have been
formulated. It is suggested that proliferation and diversification of NR sequences have continued through
nematode evolution, with distinct NRs contributing to
specific adaptations for particular lifestyles (Sluder et
al., 1999, see review Van Gilst et al., 2002). It is also
postulated, for several receptors, that the NRs originally evolved from proteins that mediate signals from
environmental compounds or nutrients (Yamamoto,
1997).
It is well documented that C. elegans requires sterol,
usually supplied as cholesterol, and this can be metabolized to unusual 4-alpha-methyl sterols (4MSs) (Merris et al., 2003). In addition, it has been shown that
sterols such as campesterol and stigmasterol are metabolized in C. elegans (Lozano et al., 1985). Other
studies in C. elegans show that sterols can be found
in excretory gland cell, amphid and phasmid socket
cells, spicule socket cells, pharynx and intestine, suggesting that sterols are not only required for cell membranes, but may be required as hormone precursor and/
or developmental effectors (Merris et al., 2003). Here,
we suggest that sterol metabolites might serve as ligands to NR. Recently, the finding in C. elegans that
daf-9 encodes a cytochrome P450 and daf-12 a nuclear
receptor, provides substantial evidence for hormonal
signaling by lipophilic molecules in C. elegans (Gerisch et al., 2001). Also, a xenobiotic sensing function
has been described for nhr-8 (Lindblom et al., 2001).
Thus, it is possible that some of these proteins serve
as ligand-independent transcription factors, the expression of which might be regulated by environmental
factors such as temperature, metal ions or pH (Enmark
and Gustafsson, 2000). For other C. elegans NRs, ligands may be found among the many natural chemicals and xenobiotics. The natural environment of C.
elegans and other free-living nematodes is the soilwater interface, a location in which the organisms
would be exposed to all water-soluble xenobiotics.
Thus, it is likely that these animals would have
evolved a wide variety of pathways for detection and
detoxification of xenobiotics, explaining the large
number of orphan NRs.
The development of simple animal models for high
throughput testing of the effect of multiple xenobiotics
and mixtures on gene expression is important for an
understanding of the dangers of environmental exposure. Our previous studies showed that C. elegans
genes other than those for vitellogenin are also sensitive to vertebrate steroid hormones. We observed
changes in the expression of members of the cytochrome P450 family, which typically can metabolize
steroid hormones, fatty acids, and xenobiotics, as well
EFFECTS
OF
STEROIDS
AND
HEAVY METALS
as genes associated with oxidation-reduction (such as
the glutathione-s-hydrogenase family). The present
study is an extension of the previous one; here we
describe expression changes of the NR family in C.
elegans by DNA microarray analysis after exposure to
steroid hormones (progesterone, cholesterol and estradiol) as well as cadmium. Additional information on
vitellogenin gene expression with regard to cholesterol
and cadmium exposure is presented.
MATERIALS AND METHODS
Culture of C. elegans
Adult N2 strain of C. elegans were grown in 250
ml of liquid S medium with Escherichia coli OP50/1
(grown in 2XTY media) according to Sulston and
Hodgkin (1988). Triplicate nematode cultures were exposed for 4 days to cholesterol or to different vertebrates steroids. All steroids, purchased from Fisher
Biotech (Fair Lawn, NJ), estrogen (17b-estradiol), progesterone (4-pregnene-3,20-dione) and cholesterol
(20-hydroxycholesterol) were prepared in absolute ethanol, and added to cultures in amounts necessary to
achieve final concentrations of 1025 (estradiol), 1025
and 1027 (progesterone), and 1029 (cholesterol) M in
culture. Control cultures received an equal volume of
absolute ethanol. For cadmium experiments short-term
(4 days) and long-term (7 days) exposure experiments
were performed. CdCl2 was purchased from Sigma (St
Louis, MO), and the stock solutions were prepared in
sterile water, and control cultures for these experiments
received sterile water. Two independent experiments
were performed with CdCl2. In the first experiment,
three different concentrations of CdCl2 were tested 1,
10 and 100 mM. In the second experiment, due to the
high toxicity observed with 100 mM of CdCl2 we tested 0.1, 1 and 10 mM CdCl2. For all the different treatments, cultured nematodes were harvested after 4 (or
7) days treatment, washed and stored at 2708C pending analysis.
RNA isolation
Total RNA was isolated from C. elegans liquid cultures using Trizol reagent following a modified method
of the protocol specified by the manufacturer (Life
Technologies, Rockville, MD). Adult worm populations were suspended in Trizol, 4 ml/ml of compacted
worms. The RNA was separated using chloroform,
precipitated with isopropyl alcohol and washed with
75% ethanol. The RNA pellet was dried under vacuum
for 5–10 minutes and dissolved in DEPC-treated water.
The poly (A1) RNA was subsequently isolated using
Separator Kit (Clontech, Palo Alto, CA) according to
the manufacturer’s instructions.
DNA microarray preparation and analysis
Poly (A1) mRNA was sent to the Stanford Microarray Database (SMD; http://genome-www.stanford.edu/
microarray/; Gollub et al., 2003), for DNA microarray
hybridization and data analysis. The microarray data
was analyzed in order to identify gene expression af-
ON
CAENORHABDITIS ELEGANS
63
fected by hormone and cadmium treatments. The normalized values used were: G/R ratio .2.6 for up-regulation and R/G ratio .2.6 for down-regulation. G:
green color that corresponds to the treated samples,
and R: red color, corresponding to the control sample.
Data were filtered for spot flag 5 0, regression correlation .0.6, and Ch1 Net (red) and Ch2 Normalized
Net (green) 5 150 to eliminate either flagged spots or
if the PCR did not work (no band or a doublet), or if
there was something abnormal about the hybridization.
Yolk protein extraction
Compacted worms were homogenized using TrisHCl buffer according to Sharrock (1983). Yolk proteins were extracted from the homogenates using the
homogenizing buffer plus 0.1% (W/V) NP-40. Protein
concentration was determined by Bradford assay using
bovine serum albumin (Calbiochem, La Jolla, CA) as
standard (Bradford, 1976), and 20 mg total protein was
loaded for each treatment group.
Electrophoresis and western blotting analysis
Yolk protein extracts were resolved in a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970). Proteins were
transferred to nitrocellulose membrane using a minitrans-blot (Bio-Rad, Hercules, CA). For immunodetection we used, as primary antibodies, anti-YP-88, and
YP-170 antibodies kindly provided by Dr. Thomas
Blumenthal, and as a secondary antibody an anti-rat
IgG conjugated to alkaline phosphatase (Sigma, St.
Louis, MO). The membranes were developed using
NBT-BCIP substrate system (Promega, Madison, WI).
Gene annotation
Different databases of C. elegans genetic information exist. We preferentially used the database
WormPD and Gene Ontology to make the annotations
for biological function and expression of the genes
studied (https://www.incyte.com/proteome/WormPD;
www.geneontology.org, respectively). WormPD is a
sub-library of the BioKnowledge Library created and
run by Proteome, Inc. In WormPD, the genes of C.
elegans are categorized according to cellular roles and
the functions of corresponding proteins. For each gene,
there is information about predicted and experimental
biological and molecular function, regulation, sequence, GenBank accession number and expression
linked to references to original research papers.
Hierarchical clustering and gene classification for
CdCl2 experiments
The raw data were uploaded into the Stanford Microarray Database (SMD). Normalized data (log
(base2) (control/experimental)) were downloaded using the following filter criteria: flag 5 0 and red or
green intensity .2.5 fold of the background intensity,
regression correlation .0.6, and the channel intensity
of red or green channel were .350. Using these criteria 19838 SUIDS (unique identifying number within
64
A. NOVILLO
TABLE 1. Summary of changes expression of NR genes examined
after exposure of cultures of C. elegans to steroid hormones and
cholesterol. The criteria used are described as follows: normalized
values used were G/R ratio . 2.6 for up-regulation and R/G ratio
. 2.6 for down-regulation. Data were filtered for spot flag 5 0,
regression correlation . 0.6, and Ch1 Net (red) and Ch2 Normalized Net (green) 5 150 to eliminate either flagged spots or errors
in the PCR. Annotations of genes for Table 1 are from WormPD
database and original research papers.
Gene name
Expression
Fold
change
Progesterone (1027 M)
nhr-69
Gut, hypodermis, uterus1
nhr-105
nhr-35
nhr-94
F48G7.11
nhr-63
R07B7.16
C06B8.1
R11G11.1*
nhr-12
4
3.6
2.9
2.6
3.9
3.4
3.1
2.7
2.8
2.7
⇑
⇑
⇑
⇑
⇓
⇓
⇓
⇓
⇓
⇓
Estradiol (1025 M)
nhr-47
Unc-55
L2 and L3, some neurons, muscles2
nhr-59
C49D10.9
R11G11.12
K12H6.1
C54F6.8
ZK1037.4
F59E11.8
TO7C5.3
C33G8.9
3.4
2.6
2.6
3
3
4.5
3.6
3.1
3.3
2.6
2.6
⇑
⇓
⇓
⇓
⇓
⇓
⇓
⇓
⇓
⇓
⇓
Cholesterol (1029 M)
nhr-66
Seam cells, neurons3
F10G2.9
nhr-68
K06B4.8
2.4
2.6
2.9
3.4
⇑
⇑
⇓
⇓
* progesterone 1025 M. Fold change respect to control is indicated
with arrows (up-regulation and down-regulation).
1 Gissendanner et al. 2004; 2 Zhou and Whaltall 1998; 3 Miyabayashi et al. 1999.
the SMD which is specific for a single arrayed clone
or PCR-amplified region of genomic DNA) passed filters. The data value-based filter used was: cutoff (only
select genes whose is absolute value .2 at least 1 array); so filter removed 19823 SUIDS. The data quality
filter (only using genes with .80% good data) removed 4 SUIDS. With this number of genes we did
hierarchical clustering followed by visual inspection to
subdivide the nodes. Most gene annotations are from
(WormPD:
https://www.incyte.com/proteome/
WormPD) and are manually annotated.
RESULTS AND DISCUSSION
Changes in NRs gene expression (Table 1)
The complete list of genes from NRs family in C.
elegans that show alterations of gene expression after
exposure to different steroids and cholesterol is presented in Table 1. To define an alteration in gene expression we used normalized values: G/R ratio .2.6
for up-regulation and R/G ratio .2.6 for down-regu-
ET AL.
lation. Using these criteria, 25 NRs genes were identified, representing 9% of total NR genes. Having defined the NRs list of genes that are altered after exposure to vertebrate steroids and cadmium, we subdivided them into genes that appeared to be
‘‘steroid-specific.’’ It is of interest that each steroid
activated or inhibited a totally different subset of
genes, therefore no overlap was observed between the
NR genes that had altered expression associated with
different steroid treatments. Of 25 NR genes, expression of 11 was altered after estradiol exposure, 10 after
progesterone and 4 after cholesterol.
Using more relaxed cut off criteria (,2 fold
change), we are able to identify 2 members of NRs
family that were down regulated after cadmium exposure. These two NRs belong to the group of nhr-58
(T13F3.2 and nhr-58). Cadmium has been shown to
influence transcription of several vertebrate nuclear receptors previously (Simons et al., 1990; Stoica et al.,
2000), and is therefore likely to influence the biological properties of these proteins.
NRs genes regulated by estrogen
Previous studies have shown that exogenous estrogen can induce vitellogenin mRNA levels (Custodia et
al., 2001; Kohra et al., 1999) in C. elegans, and at
concentration of 5 mM stimulated growth, having a
cholesterol-like potency for C. elegans (Tominaga et
al., 2001). In our study, 10 mM estradiol induced overexpression of the nhr-47 member of the NR family
(Table 1). By gene ontology, of the under-expressed
genes, the majority is predicted to be associated with
transcriptional regulation, and others are involved in
development and lipid storage. It is of interest to speculate that these genes may be part of an estrogen sensitive gene network related to vitellogenesis, since vitellogenin is dependant upon the availability of lipid
stores to the gastrointestinal cells involved in vtg synthesis. One of these NR genes (unc-55) has an important role in neurogenesis. Specifically unc-55 is implicated in neural differentiation and control of post-embryonic remodeling of the synaptic specificity of particular motor neurons (Zhou and Walthall, 1998) in C.
elegans. In the absence of unc-55 function, animals
exhibit locomotion defects due to defects in the synaptic connections of the VD motor neurons (Walthall,
1990). The unc-55 NR gene is a member of the conserved COUP NR group (NR2F). It is of interest that
vertebrate members of these families of NR are implicated in neurogenesis and regulation of eye and neural development (Fjose et al., 1993; Pereira et al.,
1995). This suggests that neural specification could be
an ancient function of this particular NR group. A role
of estrogen in vertebrate neurogenesis is an area of
intense research and significance.
NRs genes regulated by progesterone
The two different concentrations of progesterone
tested (0.1 and 10 mM) in cultures of C. elegans provoked over-expression of 4 NRs and down-regulation
EFFECTS
OF
STEROIDS
AND
HEAVY METALS
ON
CAENORHABDITIS ELEGANS
65
tebrate steroids, and (c) cholesterol as an important
component of egg (oocyte) yolk.
FIG. 1. Overview of gene expression changes in response to different doses of cadmium chloride (CdCl2: 0.1, 1, 10 mM) and cholesterol (Ch: 1029 M). The histogram shows the numbers of genes
that are repressed or induced a least twofold following criteria explained in Material and Methods.
of 6 of NRs family (Table 1). The effect on NRs
mRNA expression was not dose related, because only
one of the NR (R11G11.1, Table 1) was down regulated by high doses of progesterone (10 mM). Of particular interest, nhr-69 (up-regulated by progesterone,
Table 1) is a member of the conserved NR2A group
(the human paralog is HNF4), and is expressed in gut,
hypodermis and uterus (Gissendanner et al., 2004).
Vertebrate members of this NR family (NR2A) are involved in cholesterol and amino acid metabolism, as
well as aspects of carbohydrate, lipid and xenobiotic
metabolism, as well as other liver specific genes (Giguere, 1999). In nematodes the gut exerts many of the
functions of vertebrate hepatic tissues, such as vitellogenesis.
Response of C. elegans to cholesterol
C. elegans requires small amounts of exogenous
cholesterol for growth and as a structural component
of membranes and key metabolic intermediates. It is
also suggested that its availability has an important
role in molting and induction of a specialized nonfeeding larval stage (see review in Kurzchalia and
Ward, 2003). Supporting an important role, in this
study we show that low doses of cholesterol provoke
changes in expression of a large number of genes (Fig.
1) reflecting potential interference in a wide range of
pathways. In more detail, we will focus on genes related to lipid metabolism, transport, storage and regulation of transcription (Tables 1 and 2). Of particular
interest to the biology of C. elegans, as well as its use
as an environmental bioindicator species, are genes
implicated in: (a) regulation of transcription such as
NR and other transcription factors, (b) cholesterol metabolism to steroid signaling molecules similar to ver-
(a) Genes implicated in transcription (NRs): We
show that low concentrations of cholesterol (1 nM)
changed the expression patterns of 4 members of NRs
in C. elegans. These are nhr-66, F10G2.9, nhr-68 and
K06B4.8. The spatial and temporal expression patterns
of nhr-66 have been described by examining transgenic animals (Miyabayashi et al., 1999), showing that it
is expressed in neuronal and non-neuronal cells (hypodermal seam cells). No expression pattern has been
described for the other three regulated NR members:
nhr-68, K06B4.8, F10G2.9 but it is predicted that they
are implicated in biological processes such as steroid
metabolism, neurogenesis, and transcription (see
WormPD database). In this study, nhr-68 and K06B4.8
are down regulated by cholesterol. Studies using RNAi
showed that nhr-68 increased body fat in C. elegans
(Ashrafi et al., 2003) and K06B4.8 was implicated in
regulation of growth (Simmer et al., 2003).
In addition, cholesterol treatment caused over-expression of several transcription factors (Table 2). By
gene ontology and sequence comparison, these genes
are predicted to have diverse functions in morphogenesis, lipid storage and vulval development, reflecting
the wide range of pathways in which cholesterol is
involved. Of special importance are the changes in expression of G-protein-coupled receptors (GPCRs) suggesting that cholesterol may be involved in neuroendocrine pathways in C. elegans.
(b) Potential genes implicated in cholesterol metabolism to steroid signaling molecules similar to vertebrate steroids: Treatment of C. elegans with low concentrations of cholesterol provoked over-expression of
two genes of the P450 family: K10D2.6 and H02I12.8,
and down regulation of gst genes (Table 2). Cytochrome P450 enzymes are monooxygenases that metabolize many endogenous and exogenous lipophilic
compounds including steroid hormones, xenobiotics
and fatty acids (Mansuy, 1998). These cytochrome
P450’s may participate in synthesizing, modifying or
degrading putative hormonal derivatives of cholesterol. Although there is evidence that cholesterol metabolites, steroid hormones or ecdysones can serve as candidate ligands for nematode NR (see review Kurzchalia and Ward, 2003), there is no clear chemical
identification of these molecules in worms. Previous
studies (Custodia et al., 2001), demonstrated that C.
elegans respond to vertebrate steroids by altering expression patterns of vitellogenin, cyp450, gst and hsp
genes. In particular, gst-4 has been shown to be down
regulated by progesterone and estradiol in our previous
studies, and in this study by cholesterol. Further, we
show here that the expression of certain members of
NRs in C. elegans change after exposure to vertebrate
steroids and cholesterol, suggesting that NRs respond
to a wide variety of exogenous compounds. In this
regard, it is of interest to speculate that nutrient cholesterol can be metabolized and serves as a precursor
66
A. NOVILLO
ET AL.
TABLE 2. Summary of genes that are potentially implicated in hormonal signaling pathways in C. elegans after cholesterol treatment (1029
M). The criteria used to select these genes is as follows: normalized values used were G/R ratio . 2.6 for up-regulation and R/G ratio . 2.6
for down-regulation. Data were filtered for spot flag 5 0, regression correlation . 0.6, and Ch1 net (red) and Ch2 normalized net (green)
5 150 to eliminate either flagged spots or errors in the PCR. Annotations of genes for Table 2 are from WormPD database and original
scientific research article. Fold change with respect to control is indicated with arrows (up-regulation and down-regulation).
Gene name
Metabolic enzyme
K10D2.6
H02I12.8
B0365.1
F28H7.3
K08B12.1
gst-4
C54D10.1
Molecular and biological function
Positive regulation growth1
Embryogenesis, Energy metabolism1,2
Cholesterol transport: vitellogenin family
vit-2
Cholesterol and binding transport3
vit-3
Unknown
vit-4
vit-5
Lipoprotein oxidation, life span6,7
Lipid storage and binding
C32D5.11
W02D3.5
Lipid storage9
Transcription factor
Y5F2A.4
pos-1
dpl-1
F35H8.3
W02C12.3
Embryonic development10
Regulation cell cycle11
Transporter
ZK829.9
F56C9.3
T28F3.3
ptr-2
Expression
Intestine, oocytes4,5
Intestine8
2.9
2.5
2.9
6.5
3.9
3.8
3
⇑
⇑
⇑
⇓
⇓
⇓
⇓
3.3
9.1
4.8
2.6
⇑
⇑
⇑
⇑
2.7 ⇑
6.6 ⇓
Lipid storage9
3.4
3.3
3
2.6
2.5
⇑
⇑
⇑
⇑
⇑
Embryogenesis, Energy metabolism2
3.2
3.1
2.9
2.9
⇑
⇑
⇑
⇑
Gonad10
Ubiquitious11
HSP family
sip-1
hsp-4
F44E5.4
Receptor (GPCR family)
mes-4
Y47G7B.1
Fold change
2.6 ⇑
2.6 ⇑
4.1 ⇓
Germ cell development12
Unknown
Protein degradation
F28A12.4
Germ cell13
3.3 ⇑
2.5 ⇑
7.4 ⇓
1
Simmer et al. 2003; 2 Piano et al. 2002; 3 Matyash et al. 2001; 4 Grant and Hirsh 1999; 5 Meissner et al. 2004; 6 Shibata et al. 2003;
7 Murphy et al. 2003; 8 Kimble and Sharrock 1983; 9 Ashrafi et al. 2003; 10 Hanazawa et al. 2001; 11 Ceol and Horvitz 2001; 12 Garvin et al.
1998; 13 Fong et al. 2002.
for ligands that bind to NR (orphan receptors, OR) in
C. elegans as in vertebrates. In vertebrates, OR are
considered potential lipid sensors (Chawla et al.,
2001). In C. elegans, indirect evidence of a sterol metabolic pathway exists. Thus, blocking sterol metabolism by 25-azacoprostane-HCl treatments causes serious defects in germ cell development, growth, cuticle
development and motility (Choi et al., 2003). However, while cholesterol-derived steroids have not yet
been identified in C. elegans, the genome of C. elegans
contains approximately 80 cytochrome P450s and 17
estradiol dehydrogenases that are candidate enzymes
for modification of cholesterol to form steroid hormones (Mansuy, 1998; Nelson, 1998). It is possible
that these vertebrate OR lipid sensors represent part of
a conserved network of genes which were organized
at the outset of prokaryotic cellular organization
(around 950 Myr before present). Thus, ligand binding
to the receptor may activate a metabolic cascade (gene
network) that maintains nutrient homeostasis and all
subsequent metabolic pathways.
Studies involving the biological effects of vertebrate
steroids upon parasitic nematodes have been performed, and observations from these studies indicate
that nematodes are responsive to vertebrate host steroids. These studies indicate that vertebrate steroids
affect reproduction, growth, molting, feeding, embryogenesis and movement (reviewed in Chitwood, 1999),
suggesting the existence of a hormonal signaling pathway.
(c) Vitellogenin genes and others: Recent studies
of cholesterol distribution and transport indicate that
EFFECTS
OF
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AND
HEAVY METALS
the process of cholesterol transport in C. elegans (Matyash et al., 2001) has similarities to that of vertebrates
particularly with regard to the overall process of vitellogenesis (see Duggan et al., 2001). The studies provide support for the concept that macromolecular
transport of cholesterol in association with triglycerides, phospholipids and proteins may have co-evolved
in association with the process of oocyte yolk deposition. Vitellogenins are a primary macromolecular
transporter of cholesterol to the oocyte in C. elegans
(Matyash et al., 2001). In contrast, in vertebrates vitellogenin per se does not transport cholesterol to the
oocyte, this function being provided by the well-described LDL pathway (Brown and Goldstein, 1986),
which also delivers cholesterol to somatic cells that do
not take up vitellogenin. Non-mammalian vertebrate
oocytes express both the vitellogenin receptor (Bujo et
al., 1994) and the LDL-receptor. The argument that
vitellogenin is a primordial macromolecular transporter of cholesterol is strengthened by observations of the
homology between the primary protein of the LDL
particle (apolipoprotein B) and vitellogenin (Baker,
1988; Perez et al., 1991; Mann et al., 1999). Clearly,
other mechanisms for cellular cholesterol accumulation, such as the LDL pathway, occur in C. elegans,
because cholesterol uptake starts before yolk protein
expression, and males do not express vitellogenin but
accumulate cholesterol in sperm (Matyash et al.,
2001). In connection with this, Matyash et al. (2001)
identified a 37 KDa cholesterol binding protein in
males and hermaphrodites which is a candidate cholesterol transporter. The studies of Matyash et al.
(2001) have also shown that cholesterol is accumulated in the pharynx, nerve ring, excretory gland cell and
gut of L1–L3 larvae and later in oocytes and sperm.
It is of interest that in previous studies (Custodia et
al., 2001), using western-blot analysis we showed that
low doses of 20-hydroxycholesterol (1 nM) increased
expression of yolk proteins YP-170s. This suggests
that substrate availability (cholesterol) may influence
the process of yolk protein synthesis. In the present
study, DNA microarrays confirmed up-regulation of
the majority of members of vtg gene family (Table 2),
including vit-3 (Table 2).
In addition, one oocyte-enriched gene was up regulated after cholesterol treatment, ptr-2 (Table 2).
Studies using RNAi showed that ptr-2 has an important role in embryogenesis, embryonic cleavage, and
energy metabolism in C. elegans (Piano et al., 2002).
Response of C. elegans to cadmium
In two experiments, C. elegans cultures were exposed to CdCl2. In experiment 1, changes in gene expression were analyzed by cDNA microarrays and vitellogenin protein levels by western blot, after 0.1, 1
and 10 mM CdCl2 exposure for 7 days. No changes in
vitellogenin protein expression (data not shown) were
seen as assessed by western-blot analysis. In experiment 2, cultures of C. elegans were exposed to 1, 10
and 100 mM CdCl2 for 4 days and changes of yolk
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CAENORHABDITIS ELEGANS
67
FIG. 2. Western-blot analysis of the effect of CdCl2 on C. elegans
yolk proteins YP170, YP115 and YP88. C. elegans liquid cultures
were treated with different concentrations of CdCl2 (1, 10 and 100
mM) for four days. Then yolk protein was extracted and westernblotting was performed using a yolk protein antibody specific to
YP170s, YP115 and YP88. C: control group.
protein expression were assessed by western blot analysis (Fig. 2); microarray analysis was not done for this
experiment. By western-blot analysis the expected
number and size of vitellogenin translation products
were detected in both experiments. High concentrations of cadmium (100 mM CdCl2) resulted in inhibition of translation of the three vitellogenin proteins
(Fig. 2), and clear toxicity effects were observed (mortality . 70%), suggesting the inhibition observed in
vitellogenin protein levels is due to toxicity. By western blot analysis, exposure to 10 mM CdCl2 resulted
in a slight inhibition of yolk proteins YP170s and
YP115, but was without effect on YP88.
The differences in observed vitellogenin protein levels in response to cadmium in the two different experiments may be explained by differences in exposure
time (4 day versus 7 day), and the high levels of expression of mtl-2 and cdr-1 genes (see below) observed in experiment 1. Expression of these genes
would protect the organism from cadmium toxicity,
and prevent the inhibition of vitellogenin gene expression observed when exposed to cadmium for a longer
time. However, microarray analysis will be necessary
to assess this possibility. In support of a protective role
of metallothionein gene expression and cadmium sequestration in vitellogenin gene expression, de novo
induction of vitellogenin synthesis has been shown to
occur in the rainbow trout once metallothionein has
begun to sequester cadmium (Olsson, 1995).
Cadmium triggered expression changes in a small
number of genes in a dose dependant manner (Fig. 1).
To summarize the data for microarrays and the changes
in gene expression induced by cadmium treatment, we
used a hierarchical clustering algorithm to sort the
genes according to their similarity in expression pattern during the exposure time to different doses of
CdCl2. The hierarchical clustering sorted the genes into
two main nodes (Fig. 3). Almost all the genes in the
first node (cluster A) are over-expressed in relation to
the dose of cadmium, while almost all the genes in the
second node (cluster B) are under-expressed in relation
68
A. NOVILLO
FIG. 3. Hierarchical clustering was used to display the expression
ratios of the cadmium-responsive genes in C. elegans treated cultures.
to CdCl2 exposure. 50% of all the genes in the first
node (cluster A) have peak over-expression in adults
in the 10 mM CdCl2 treatment, and the other 50% of
the genes have almost the same level of gene expression irrespective of the concentration of cadmium used
in the experiment.
To identify cellular functions that can be affected by
cadmium treatment, we determined which functional
classes of genes were over-expressed in this study (Fig.
4). Thus, up regulated genes were placed into one of
the following putative molecular function classes: cell
communication, stress signaling, GPCR receptors, metabolism, immunoglobulin signaling, collagens, detoxification; genes which could not be assigned to a functional class were placed into the Unknown class.
In more detail, to protect against cadmium-induced
damage, cells respond by increasing the expression of
genes encoding stress-responsive proteins implicated
in detoxification. In this study, two genes that are specific for the cadmium-induced response have been
identified: mtl-2 (member of metallothionein family)
and cdr-1. The ability of cadmium to induce metallothionein gene expression in a variety of species has
been documented (Liao and Freedman, 1998; Waisberg et al., 2003) and a detailed discussion of the effect of cadmium on C. elegans metallothionein gene
transcription can be found in Freedman et al. (1993).
The other gene, cdr-1, recently identified, is a cadmium inducible lysosomal protein required for resistance
to cadmium (Liao et al., 2002). These two genes are
expressed in intestine, and are transcriptionally regulated by cadmium through a specific metal-responsive
element (MRE) found in the promoter region of both
genes. High levels of expression of these two genes
suggest the possibility that C. elegans can survive and
avoid the negative effects of cadmium by sequestration
of metal ions. In addition, other genes that respond to
metal stress have been identified (Fig. 4). These are:
Y73F8A.H, Y1055A.C, Y105C5A.D, F19G12.7; according to their predicted function these genes are involved in repair processes after stress (based on sequence similarity, WormPD). The mechanism by
ET AL.
FIG. 4. Relative representation of genes with different molecular
functions that are up-regulated in this study after cadmium treatment.
Genes were placed into one of the following putative molecular
functional classes as described in Methods: cell communication,
stress signalling, GPCR receptors, metabolism, immunoglobulin signaling, collagens, detoxification; genes which could not be assigned
to a functional class were placed into the Unknown class (Unknown). Shown are % of genes in each functional class respect to
the total of genes up regulated with cadmium.
which cadmium affects the expression of these genes
remains unknown, but possibly involves the presence
of MREs in promoter regions of these genes.
An important functional class of genes which are
related to cuticle formation and collagen are over-expressed after cadmium exposure. The effect of cadmium on collagen genes is well documented by both
in vivo and in vitro studies. Cadmium acts as a profibrinogenic agent in liver, and it is suggested that oxidative stress may stimulate lipid peroxidation and collagen synthesis (del Carmen et al., 2002; Liao and
Freedman, 1998).
We also observed that cadmium induced over-expression of C09H5.2 gene, which is a member of the
E1–E2 ATPase family. This predicted gene, according
to sequence similarity and domain content, may be implicated in cation transport and metal ion homeostasis.
Cadmium may displace metal ions from proteins by
altering the homeostasis of metals such as Zn and Ca,
possibly explaining the effect of cadmium on gene expression. In turn, signal transduction pathways are impacted which then can influence the expression of myriad genes. 50% of genes over-expressed after cadmium
treatment have unknown functions (Fig. 4). Of these,
many have ubiquitin transferase domain that is involved in the degradation of unfolded proteins.
Relevance to endocrine disruption
C. elegans has been used as biosensor in environmental monitoring studies since the late 1980s (see
review in Custodia et al., 2001). Previous studies from
this laboratory demonstrated that C. elegans responds
to vertebrate steroids by altering expression patterns of
vitellogenin, cyp450, gst and hsp genes suggesting that
this organism may be useful laboratory model for
screening of endocrine disruptor compounds (EDCs)
(Custodia et al., 2001). In this study, we show that the
expression of certain members of NRs in C. elegans
change after exposure to vertebrate steroids and cholesterol, suggesting that NRs can respond to a variety
EFFECTS
OF
STEROIDS
AND
HEAVY METALS
of exogenous steroids. This may influence the regulation of metabolic and developmental pathways, allowing nematodes to adapt and exploit changing environments.
That steroids, particularly estrogen, widely impact
gene expression in C. elegans suggest that this model
may be used to assess the actions of xenoestrogens and
other environmental contaminants in the laboratory at
the genome level. In this regard, cadmium, as a representative of the toxic heavy metal group, is of particular interest, as it may act in an estrogen-like manner. Cadmium is a heavy metal with no known biological function and it is one of the more serious environmental pollutants. It has been classified as a
group 1 human carcinogen (IARC, 1993) and it is listed by the US EPA as one of 126 priority pollutants.
Recently it has been reported that cadmium can provoke direct inhibition of DNA mismatch repair (Jin et
al., 2003), thus cadmium toxicity can represent a new
mechanism by which genomes can be destabilized
with profound implications for human health, risk assessment and biological understanding of environmental mutagens (see review McMurray and Tainer, 2003).
Furthermore, it has been shown that cadmium has potent estrogen-like activity in vivo (Johnson et al.,
2003), adding a new dimension to currently used riskassessment protocols for this environmental chemical.
In vivo and in vitro studies have shown that dysregulation of gene expression is a major factor that can
explain the multiple effects of cadmium exposure. A
survey of genes that are induced by cadmium (reviewed by Waisberg et al., 2003), classifies them into
four different groups: (1) Immediate early response
genes (IEGs) such as c-fos, p53, c-jun; (2) Stress response genes such as mtl, hsp, and genes controlling
glutathione and related proteins; (3) transcription and
translation factors; (4) miscellaneous genes such as
collagen, rRNAs, pyruvate carboxylase. Thus, many
cellular processes can be disrupted by this heavy metal.
C. elegans is a powerful animal model for the study
of functional genomics (see review Kim, 2001). DNA
microarray experiments can be used to determine expression changes after different exposures, in different
mutants with different reproductive backgrounds to
provide new insights into invertebrate and vertebrate
biology. A combination of microarray and functional
data from web-based databases will make it possible
to analyze gene network pathways implicated in endocrine disruption. It will be important to develop C.
elegans and other species such as Danio rerio into
high through-put models in which a wide variety of
xenobiotics may be tested in low concentrations for
genome wide-responses. Comparative genomic studies
have shown that 83% of C. elegans genes have human
homologs (Lai et al., 2000), thus such studies will aid
in our comprehension of the impact of xenobiotics on
the human genome. Beyond direct implications for human biology and health, to understand the effects of
EDC on reproduction and development is of cardinal
ON
CAENORHABDITIS ELEGANS
69
importance for maintenance of biodiversity and invertebrate species, which comprise approximately 95% of
all terrestrial and aquatic animal species (Wilson,
1999).
ACKNOWLEDGMENTS
This study was Supported by NIH ES 07381 to IPC.
Apolonia Novillo was supported by a postdoctoral fellowship from Ministry of Science and Technology of
Spain.
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